Dr Piyush Tailor

1. PURPOSE OF EXAMINATION: To lay down the procedure for Alcian Blue staining in Histo-pathology section at NCHLS Surat.

2. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION: Alcian blue is a group of polyvalent cationic dye that is water soluble. It forms reversible electrostatic bond between cations of the dye and anions of polysaccharide molecule. Alcian blue at 2.5 pH stains carboxylated acid mucopolysaccharide and at pH 1 sulaphated mucin are stain. Blue color is due to presence of copper in molecule. It is believed that dye forms salt linkage with acid groups of polysaccharides.

3. STAFF RESPONSIBLE: lab. technician under supervision of resident doctor/tutor.

4. PERFORMANCE CHARACTERISTICS: NA

5. TYPE OF SAMPLE: Histopathology section slide

6. PATIENTS PREPARATIONS: NA

7. TYPE OF CONTAINER AND ADDITIVES:

8. POSITIVE CONTROL: Small intestine, Appendix, Colon.

9. REQUIRED EQUIPMENTS AND REAGENTS:

EQUIPMENTS: Coplin jars, Glasswares, Oven

REAGENT: Alcian blue, Acetic Acid, Thymol, Harris hematoxylin, absolute alcohol, Distilled water, Xylene, DPX.

10. ENVIRONMENTAL AND SAFETY CONTROLS: Universal safety precautions

11. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):NA 12. PROCEDURE(STEPS):

Alcian blue solution preparation-

Boil 100ml distilled water and add 3ml of concentrated acetic acid to it. Add 1gm of Alcian blue (8GX DYE). It takes lot of time to dissolve Alcian blue dye. Adjust ph to 2.5, using acetic acid. Add a crystal of thymol.

Staining Procedure:

• Take test slide on which staining has to be performed along with positive control.

• Deparaffinize section by keeping in oven for 5min and clear it with 2 changes of xylene each for 15 minute xylene.

• Dehydrate section with graded alcohol 2 changes each for 2 minutes.

• Bring section to distilled water.

• Stain in freshly prepared 1% Alcian blue for 30 minute.

• Wash in tap water wash for 2 min.

• Dehydrate in alcohol, clear in xylene, mount with DPX

Result

• Nuclei : Pale blue

• Acid mucin- Deep blue

13. QUALITY CONTROL PROCEDURE: The newer batch of stain is tested before being put in to use by checking their efficiency with the batch of stain already in use.

14. INTERFERENCE: Inadequate quality/improper dilution

15. PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING, WHERE RELEVENT, THE MEASUREMENT OF UNCERTAINITY OF MEASURED QUANTITY VALUES: NA

16. BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES:NA

17. REPORTABLE INTERVAL OF EXAMINATION RESULTS: NA

18. INSTRUCTION FOR DETERMINING QUANTITATIVE RESULTS WHEN RESULTS IS NOT WITHIN THE MEASUREMENT INTERVAL: NA

19. ALERT /CRITICAL VALUE, WHERE APPROPRIATE: NA

20. LABORATORY CLINICAL INTERPRETATION: Interpret test result after true positivity of control slide.

21. POTENTIAL SOURCE OF VARIATION: NA

22. REFERENCE: Histopathological laboratory techniques by Culling’s.